Assays for detection of Bacillus anthracis

ABSTRACT

This invention provides novel methods, reagents, and kits that are useful for detecting  B. anthracis . The methods are based on the discovery of binding agents, including recombinant polyclonal antibodies, which bind to the surface array protein of  B. anthracis.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 09/754,947, filed Jan. 4, 2001, now U.S. Pat. No. 6,828,110,issued Dec. 7, 2004, which claims benefit of priority to U.S.Provisional Patent Application No. 60/174,901, filed Jan. 6, 2000, eachof which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention pertains to the field of assays for detecting Bacillusanthracis, the causative agent of anthrax.

BACKGROUND

Anthrax spores were first produced as weapons in the 1950s. Severalcountries including the former Soviet Union, the United States and. Iraqare known to have produced anthrax weapons. Anthrax is a particularlyfearsome biological warfare agent, not only because of its deadliness,but also because anthrax weapons are relatively easy to produce anddeliver. Production of anthrax spores requires little more than basiclaboratory equipment and growth media. Anthrax weapons are comprised ofan anthrax source and an industrial sprayer that can produce aerosolparticles of the appropriate size for victims to inhale. Such sprayers,for instance, can be mounted on low flying airplanes or other vehiclesand used to spread anthrax over a wide area. Because of the ease andrelatively small expense involved in producing and delivering anthraxweapons, such weapons are potentially highly attractive weapons of massdestruction for terrorist groups. Thus, in addition to potentialorganized military conflicts that may give rise to the use of suchweapons, terrorist organizations are a potential threat for the use ofsuch weapons in airports, office buildings and other centers of humanactivity.

Anthrax is caused by Bacillus anthracis, a gram-positive, sporulatingbacillus. B. anthracis is a soil bacterium and is distributed worldwide.The organism exists in the infected host as a vegetative bacillus and inthe environment as a spore. The anthrax spore is typically the infectiveform of the bacterial life cycle. Anthrax spores can survive adverseenvironmental conditions and can remain viable for decades. Animals suchas cattle, sheep, goats and horses can contract the spores whilegrazing. Humans can contract anthrax from inoculation of minor skinlesions with spores from infected animals, their hides, wool or otherproducts, such as infected meat (Franz et al. (1997) J. Am. Med. Assoc.278(5): 399-411).

The typical mode of entry of the anthrax spore into the body,inhalation, results in an illness known as woolsorter's disease. Afterdeposit in the lower respiratory tract, spores are phagocytized bytissue macrophages and transported to hilar and mediastinal lymph nodes.The spores germinate into vegetative bacilli, producing a necrotizinghemorrhagic mediastinitis (Franz et al., supra). Symptoms include fever,malaise and fatigue, which can easily be confused with the flu. Thedisease may progress to an abrupt onset of severe respiratory distresswith dyspnea, stridor, diaphoresis and cyanosis. Death usually followswithin 24 to 36 hours.

Because the effects of exposure to anthrax are not immediate, andbecause the initial symptoms are easily confused with the flu, there isa need for a fast method to detect B. anthracis in an environment whereB. anthracis may have been released. This need is enhanced by theincreasing number of anthrax threats that are called into governmentalauthorities each year. A fast method for determining whether publicplaces have been exposed to anthrax spores in therefore essential.

Anthrax spores have S-layers, as do spores of many other archea andbacteria. Most S-layers are comprised of repeats of a single protein(Etienne-Toumelin et al., J. Bacteriol. 177:614-20 (1995)). The S-layerof B. anthracis, however, is comprised of at least two proteins: EA1(Mesnage et al., Molec. Microbiol. 23:1147-55 (1997)) and surface arrayprotein (SAP) (see Etienne-Toumelin, et al., supra). Fully virulent B.anthracis isolates are encapsulated by a capsule that encompasses theS-layer of the bacteria and prevents access of antibodies to both EA1and SAP (Mesnage et al., J. Bacteriol. 180:52-58 (1998)).

Several methods for detecting B. anthracis have been reported, althoughnone are optimal for quick and reliable detection of anthraxcontamination. Detection methods include those based on amplification ofnucleic acids that are specific for B. anthracis (Lee, J. Appl.Microbiol. 87:218-23 (1999); Patra, G., FEMS Immunol. Med. Microbiol.15:223-31 (1996); Ramisse et al, FEMS Microbiol. Lett. 145(1):9-15(1996); Bruno and Keil, Biosens. Bioelectron. 14:457-64 (1999); andJapanese Patent Nos. 11004693; 6261759; 6253847; and 6253846). The needto conduct time-consuming laboratory procedures to use theseamplification methods limits their usefulness for quick identificationof anthrax contamination. Other detection methods involve detectingspore-based epitopes of B. anthracis using antibodies (Yu, H., J.Immunol. Methods 218:1-8 (1998); Phillips et al., J. Appl. Bacteriol.64:47-55 (1988); Phillips et al., FEMS Microbiol. Immunol. 1:169-78(1988)). Other reported detection methods include an enzyme-linkedlectinosorbent assay (Graham et al., Eur. J. Clin. Microbiol. 3:210-2(1984)) and a method using DNA aptamers that bind anthrax spores (Brunoet al., Biosens Bioelectron. 14(5):457-64 (1999)).

Previous antibody-based detection methods for B. anthracis employedantibodies raised against whole anthrax spores. Such immunogens lead tothe production of antibodies that cross-react with other relatedbacterial species. Longchamps et al., for instance, found that noantibody analyzed in their study was completely specific in recognizinganthrax spores (J. Applied Microbiology 87:246-49 (1999)). At least onestudy has shown that polyclonal antibodies raised against B. anthraciswhole spore suspensions do not react with SAP protein (Mesnage et al,Molec. Microbiol. 23:1147-55 (1997)). Closely related bacteria that maycross react with non-specific antibodies include B. cereus, B.thuringiensis and B. mycoides (Longchamp et al., supra.; Phillips etal., FEMS Microbiol. Immunol. 47:169-78 (1988)). This high degree ofcross-reactivity is highly problematic for detection of anthrax becausethese non-toxic cross-reactive strains are widespread. B. thuringiensisin particular is commonly found in the soil, in part because thebacteria is sprayed on crops for its insecticidal qualities.

Therefore, a need exists for improved methods for detecting Bacillusanthracis in the environment. Such methods should be not only providerapid results, but also should have little or no cross-reactivity withrelated species that are prevalent in nature. The present inventionfulfills this and other needs.

SUMMARY OF THE INVENTION

The present invention provides novel methods of detecting Bacillusanthracis. The methods involve contacting a test sample with a capturereagent that can bind to Bacillus anthracis surface array protein (SAP),wherein the capture reagent forms a complex with SAP if SAP is presentin the test sample, and detecting whether SAP is bound to the capturereagent. The capture reagent, for instance, can form a complex with thesurface array protein if the surface array protein is present in thesample. Presence of the surface array protein is indicative of thepresence of B. anthracis in the sample. In one embodiment, SAP comprisesa polypeptide with the amino acid sequence shown in SEQ ID NO:1. Inanother embodiment, the B. anthracis strain is encapsulated.

The capture reagent can comprise an antibody that binds to SAP. In someembodiments, the antibody can be a recombinant antibody, such as arecombinant polyclonal or monoclonal antibody.

In a preferred embodiment, the test sample is collected from a site ofsuspected or threatened anthrax contamination. In another preferredembodiment, the test sample is collected using a cyclonic device. Thetest sample does not need to be cultured prior to contacting with thecapture reagent.

In some methods of the invention, the capture reagent can be immobilizedon a solid surface, such as a microtiter dish. The capture reagent canbe immobilized on the solid support prior to contacting the capturereagent with the test sample.

In presently preferred embodiments, the assay methods of the inventionare highly sensitive. For instance, in one embodiment, antibodies of theinvention used according to the methods of the invention can detect B.anthracis at concentrations at least as low as 10,000 cfu/ml. In a morepreferred embodiment, the methods of the invention are capable ofdetecting B. anthracis at concentrations at least as low as 5,000cfu/ml. In still more preferred embodiments, the methods of theinvention are capable of detecting B. anthracis at concentrations atleast as low as 1,800 cfu/ml.

In some embodiments, SAP is detected by contacting SAP with a detectionreagent that can bind SAP. Like the capture reagent, the detectionreagent can be an antibody that binds SAP. For instance, the detectionreagent can bind a different epitope of SAP than the capture agentbinds. In some embodiments, the detection reagent comprises a detectablelabel. The detectable label can be, for instance, a radioactive label, afluorophore, a dye, an enzyme or a chemiluminescent label.

The invention also provides devices and kits for detecting B. anthracis.The kits typically include, inter alia, a solid support upon which isimmobilized a capture reagent which binds to a SAP of B. anthracis, anda detection reagent which binds to the SAP. In some embodiments thesolid support is a microtiter dish. In another embodiment, the capturereagent is an antibody, such as a recombinant polyclonal or monoclonalantibody or mixtures thereof. The kit can also include writteninstructions for using the kit to determine whether a test samplecontains B. anthracis. In some embodiments, the kit also comprises apositive control that comprises a polypeptide that comprises anantigenic determinant of B. anthracis SAP. The SAP can be, for example,the amino acid sequence displayed in SEQ ID NO:1.

The invention also provides for recombinant polyclonal antibodypreparations that specifically bind to an antigenic determinant of B.anthracis SAP. For instance, the SAP polypeptide can be the amino acidsequence displayed in SEQ ID NO:1.

DETAILED DESCRIPTION Definitions

The phrase “capture reagent” refers to a molecule that specificallybinds to a specific target molecule. For instance, the target moleculecan be a surface array protein (SAP) of Bacillus anthracis, or a portionthereof. Capture reagents include antibodies as well as naturally andnon-naturally-occurring molecules that can specifically bind a targetmolecule. For instance, peptides that specifically bind a targetmolecule and are developed using phage display or other combinatorialsystem are encompassed by this definition.

A “test sample” is a sample obtained from a non-laboratory source thatis not known to contain B. anthracis. For example, a sample grown onlaboratory growth media or purified from laboratory growth media is nota test sample unless it is not known whether the sample contains B.anthracis.

The phrases “specifically binds to” or “specifically immunoreactivewith”, when referring to an antibody or other binding moiety refers to abinding reaction which is determinative of the presence of a targetantigen in the presence of a heterogeneous population of proteins andother biologics. Thus, under designated assay conditions, the specifiedbinding moieties bind preferentially to a particular target antigen anddo not bind in a significant amount to other components present in atest sample. Specific binding to a target antigen under such conditionsmay require a binding moiety that is selected for its specificity for aparticular target antigen. A variety of immunoassay formats may be usedto select antibodies that are specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with an antigen. See Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background. Specific binding betweenan antibody or other binding agent and an antigen generally means abinding affinity of at least 10⁶ M⁻¹. Preferred binding agents bind withaffinities of at least about 10⁷ M⁻¹, and preferably 10⁸ M⁻¹ to 10⁹ M⁻¹or 10¹⁰ M⁻¹.

The term “epitope” means an antigenic determinant that is capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Epitopes can include non-contiguous amino acids, aswell as contiguous amino acids.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (See, e.g., Paul, Fundamental Immunology, 3^(rd)Ed., 1993, Raven Press, New York).

The variable regions of each light/heavy chain pair form the antibodybinding site. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarily determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. CDR and FRresidues are delineated according to the standard sequence definition ofKabat et al., supra. An alternative structural definition has beenproposed by Chothia et al. (1987) J. Mol. Biol. 196: 901-917; (1989)Nature 342: 878-883; and (1989) J. Mol. Biol. 186: 651-663.

The term “antibody” is used to mean whole antibodies and bindingfragments thereof. Binding fragments include single chain fragments, Fvfragments and Fab fragments The term Fab fragment is sometimes used inthe art to mean the binding fragment resulting from papain cleavage ofan intact antibody. The terms Fab′ and F(ab′)₂ are sometimes used in theart to refer to binding fragments of intact antibodies generated bypepsin cleavage. Here, “Fab” is used to refer generically to doublechain binding fragments of intact antibodies having at leastsubstantially complete light and heavy chain variable domains sufficientfor antigen-specific bindings, and parts of the light and heavy chainconstant regions sufficient to maintain association of the light andheavy chains. Usually, Fab fragments are formed by complexing afull-length or substantially full-length light chain with a heavy chaincomprising the variable domain and at least the CH1 domain of theconstant region.

An “isolated” species or population of species means an object species(e.g., binding polypeptides of the invention) that is the predominantspecies present (i.e., on a molar basis it is more abundant than otherspecies in the composition). Preferably, an isolated species comprisesat least about 50, 80 or 90 percent (on a molar basis) of allmacromolecular species present. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids, refers to two or more sequences or subsequences that have atleast 80%, preferably 85%, most preferably 90-95% nucleotide identity,when compared and aligned for maximum correspondence, as measured usingone of the following sequence comparison algorithms or by visualinspection. For amino acid sequences, “substantially identical” refersto two or more sequences or subsequences that have at least 60%identity, preferably 75% identity, and more preferably 90-95% identify,when compared and aligned for maximum correspondence, as measured usingone of the following sequence comparison algorithms or by visualinspection. Preferably, the substantial identity exists over a region ofthe nucleic acid or amino acid sequences that is at least about 10residues in length, more preferably over a region of at least about 20residues, and most preferably the sequences are substantially identicalover at least about 100 residues. In a most preferred embodiment, thesequences are substantially identical over the entire length of thespecified regions (e.g., coding regions).

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

A further indication that two nucleic acids or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross-reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent substitutions” or “silentvariations,” which are one species of “conservatively modifiedvariations.” Every polynucleotide sequence described herein whichencodes a polypeptide also describes every possible silent variation,except where otherwise noted. Thus, silent substitutions are an impliedfeature of every nucleic acid sequence which encodes an amino acid. Oneof skill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine and UGG, the onlycodon for tryptophan) can be modified to yield a functionally identicalmolecule by standard techniques. In some embodiments, the nucleotidesequences that encode the enzymes are preferably optimized forexpression in a particular host cell (e.g., yeast, mammalian, plant,fungal, and the like) used to produce the enzymes.

Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties are also readily identifiedas being highly similar to a particular amino acid sequence, or to aparticular nucleic acid sequence which encodes an amino acid. Suchconservatively substituted variations of any particular sequence are afeature of the present invention. Individual substitutions, deletions oradditions which alter, add or delete a single amino acid or a smallpercentage of amino acids (typically less than 5%, more typically lessthan 1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.See, e.g., Creighton (1984) Proteins, W. H. Freeman and Company.

Description of the Preferred Embodiments

The present invention provides novel kits and methods for detecting thepresence or absence of B. anthracis in a test sample. The kits andmethods are a rapid, accurate and cost-effective means for detecting B.anthracis. The methods involve, in presently preferred embodiments,contacting a test sample with a capture reagent that can bind to B.anthracis SAP. The capture reagent then forms a complex with the SAP ifit is present in the sample. The SAP is then detected to determinewhether the test sample contains B. anthracis. Typically, detection isaccomplished using a detection reagent that specifically binds to B.anthracis SAP. Both capture reagents and the detection reagentstypically use binding moieties that can bind to SAP.

Unlike previously available anthrax detection methods, the methods andkits of the invention are highly sensitive. The assays and kits, inpresently preferred embodiments, can detect B. anthracis when present ina sample at a concentration of about 10⁴ cfu/ml or less. Preferably, thedetection limit for B. anthracis will be about 5×10³ cfu/ml or less,more preferably about 1.8×10³ cfu/ml or less, and still more preferablyabout 10³ cfu/ml or less.

Moreover, the methods and kits are highly specific for B. anthracis.Unlike previously available methods, the methods and kits of the presentinvention do not suffer from cross-reactivity with non-anthraxmicroorganisms. Previous methods of detecting B. anthracis relied onantibodies raised against whole anthrax spores, so these assays sufferfrom significant cross-reactivity. In contrast, the assays of thepresent invention use binding reagents that are directed to a B.anthracis antigen that is specific for B. anthracis. This antigen issecreted and can be deposited on the surface of anthrax spores and otherparticles, for example, during the preparation of anthrax-basedbiological weapons. Thus, in addition to the high specificity of thedetection methods of the invention, the methods are more efficient andeasy to use because there is no need to disrupt the anthrax spores forbinding reagents to bind their antigens. Nor must samples suspected ofcontaining B. anthracis be cultured prior to testing.

A. Binding Moieties that Specifically Bind B. anthracis Surface ArrayProtein

The assays of the invention involve detecting the presence in a testsample of a B. anthracis SAP polypeptide, which is an antigen that isspecific for B. anthracis. The assays for detecting SAP are, in someembodiments, binding assays. In these assays, which includeimmunoassays, SAP is immobilized on a solid support using a capturereagent that can specifically bind to SAP. The immobilized SAP is thendetected using detection reagents that also are capable of specificallybinding to SAP. The detection reagents typically include at least abinding moiety and a detectable label.

The invention provides binding reagents that are capable of specificallybinding to the SAP antigen. These binding reagents can be used in one ormore steps of the assay. For example, the binding reagents can beimmobilized on a solid support and used to immobilize SAP on the solidsupport; such immobilized binding reagents are referred to herein as“capture reagents.” Binding reagents can also be used to detect B.anthracis antigens by, for example, attaching a detectable label to abinding moiety that binds to SAP. Suitable binding moieties include anymolecule that is capable of specifically binding to SAP. Antibodies andfragments thereof are examples of binding components that are suitablefor use in detection moieties.

1. Types of Binding Moieties

The invention provides binding moieties (or reagents) that canspecifically bind B. anthracis SAP polypeptides. Binding reagents canalso be, for example, antibodies prepared using as immunogens natural,recombinant or synthetic polypeptides derived from B. anthracis SAP. Theamino acid sequence of a B. anthracis SAP is shown as SEQ ID NO:1. Suchpolypeptides can function as immunogens that can be used for theproduction of monoclonal or polyclonal antibodies. Immunogenic peptidesderived from SAP can also be used as immunogens; such peptides aresometimes conjugated to a carrier polypeptide prior to inoculation.Naturally occurring, recombinantly produced, or synthetic peptides orpolypeptides are suitable for use as immunogens. These can be used ineither pure or impure form. Production of antibodies against SAPpolypeptides of the invention is discussed in more detail below.Suitable binding moieties also include those that are obtained usingmethods such as phage display.

Various procedures known in the art can be used for the production ofantibodies that specifically bind to SAP. For the production ofpolyclonal antibodies, one can use SAP to inoculate any of various hostanimals, including but not limited to rabbits, mice, rats, sheep, goats,and the like. The SAP polypeptide can be prepared by recombinant meansas described above using an expression vector containing a nucleic acidthat encodes the B. anthracis SAP. For example, a nucleotide sequenceencoding a B. anthracis SAP beginning at approximately 30 amino acidsfrom the published N-terminus (i.e., at the presumed cleavage sequence)is presented in SEQ ID NO:2.

Monoclonal antibodies can be prepared by any technique that provides forthe production of antibody molecules by continuous cell lines inculture, including the hybridoma technique originally developed byKohler and Milstein ((1975) Nature 256: 495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al. (1983)Immunology Today 4: 72), and the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al. (1985) in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonalantibodies also can be produced in germ-free animals as was described inPCT/US89/02545 (Publication No. WO8912690, published Dec. 12, 1989) andU.S. Pat. No. 5,091,512.

Fragments of antibodies are also useful as binding moieties. Whilevarious antibody fragments can be obtained by the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term “antibody,” as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv). Single chain antibodies are alsouseful to construct detection moieties. Methods for producing singlechain antibodies were described in, for example, U.S. Pat. No.4,946,778. Techniques for the construction of Fab expression librarieswere described by Huse et al. (1989) Science 246: 1275-1281; thesetechniques facilitate rapid identification of monoclonal Fab fragmentswith the desired specificity for SAP. Suitable binding moieties alsoinclude those that are obtained using methods such as phage display.

To prepare a suitable antigen preparation, one can prepare an expressionlibrary from B. anthracis and screen the library with a polyclonalantibody that is raised against a crude preparation of SAP. The insertsfrom those expression plasmids that express the SAP are then subclonedand sequenced. The SAP-encoding inserts are cloned into an expressionvector and used to transform E. coli or other suitable host cells. Theresulting preparation of recombinant SAP is then used to inoculate ananimal, e.g., a mouse.

In preferred embodiments, the binding reagents are recombinantlyproduced polyclonal or monoclonal antibodies that bind to SAP.Recombinant antibodies are typically produced by immunizing an animalwith SAP, obtaining RNA from the spleen or other antibody-expressingtissue of the animal, making cDNA, amplifying the variable domains ofthe heavy and light immunoglobulin chains, cloning the amplified DNAinto a phage display vector, infecting E. coli, expressing the phagedisplay library, and selecting those library members that express anantibody that binds to SAP. Methods suitable for carrying out each ofthese steps are described in, for example U.S. patent application Ser.No. 08/835,159, filed Apr. 4, 1997. In preferred embodiments, theantibody or other binding peptides are expressed on the cell surface ofa replicable genetic unit, such as a filamentous phage, and especiallyphage M13, Fd and F1. Most work has inserted libraries encodingpolypeptides to be displayed with either pIII or pVIII of these phage,forming a fusion protein which is displayed on the surface of the phage.See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO92/01047 (gene III); Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII).

In a preferred embodiment, the genes that encode the heavy and lightchains of antibodies present in the cDNA library are amplified using aset of primers that can amplify substantially all of the different heavyand light chains. The resulting amplified fragments that result from theamplification step are pooled and subjected to asymmetric PCR so thatonly one strand (e.g., the antisense strand) is amplified. The singlestrand products are phosphorylated, annealed to a single-stranded uraciltemplate (e.g., the vector BS45, described in U.S. patent applicationSer. No. 08/835,159, which has coding regions for the constant regionsof mouse heavy and light chains), and introduced into a uracil DNAglycosylase⁺ host cell to enrich for vectors that contain the codingsequences for heavy and light chain variable domains.

To screen for phage that express an antibody that binds to SAP, one canattach a label to SAP using methods known to those of skill in the art.In a preferred embodiment, the phage that display such antibodies areselected using SAP to which is attached an immobilizable tag, e.g.,biotin. The phage are contacted with the biotinylated antigen, afterwhich the phage are selected by contacting the resulting complex withavidin attached to a magnetic latex bead or other solid support. Theselected phage are then plated, and may be screened with SAP to which isattached a detectable label.

In a preferred embodiment, the library is enriched for those phage thatdisplay more than one antibody that binds to SAP. Methods and vectorsthat are useful for this enrichment are described in U.S. patentapplication Ser. No. 08/835,159. The panning can be repeated one or moretimes to enhance the specificity and sensitivity of the resultingantibodies. Preferably, panning is continued until the percentage offunctional positives is at least about 70%, more preferably at leastabout 80%, and most preferably at least about 90%.

A recombinant anti-SAP monoclonal antibody can then be selected byamplifying antibody-encoding DNA from individual plaques, cloning theamplified DNA into an expression vector, and expressing the antibody ina suitable host cell (e.g., E. coli). The antibodies are then tested forability to bind SAP.

Recombinant polyclonal antibodies are particularly preferred because ofthe various forms of SAP that may be found in clinical samples due to,for example, proteolysis. The diverse fine binding specificity ofmembers of a population of polyclonal antibodies often allows thepopulation to bind to several forms of SAP (e.g., species variants,escape mutant forms, proteolytic fragments) to which a monoclonalreagent may be unable to bind. Methods for producing recombinantpolyclonal antibodies are described in U.S. patent application Ser. No.08/835,159, filed Apr. 4, 1997. Specific methods of producingrecombinant polyclonal antibodies that bind to SAP are described in theExamples below.

Polyclonal antibodies can be prepared as described above, except that anindividual antibody is not selected. Rather, the pool of phage is usedfor the screening, preferably using an equal number of phage from eachsample. In preferred embodiments, the phage are enriched for those thatdisplay more than one copy of the respective antibodies. The phage arethen selected for those that bind to SAP. For example, one can use abiotinylated anti-SAP monoclonal antibody and SAP to concentrate thosephage that express antibodies that bind to SAP. The biotinylatedmonoclonal antibody is immobilized on a solid support (e.g., magneticlatex) to which is attached avidin. The phage that are bound to theimmobilized SAP are eluted, plated, and the panning repeated until thedesired percentage of functional positives is obtained.

2. Detection Reagents of the Invention

The presence of SAP is generally detected using a detection reagent thatis composed of a binding moiety that specifically binds to SAP. Suitablebinding moieties are discussed above. The detection reagents are eitherdirectly labeled, i.e., comprise or react to produce a detectable label,or are indirectly labeled, i.e., bind to a molecule that is itselflabeled with a detectable label. Labels can be directly attached to orincorporated into the detection reagent by chemical or recombinantmethods.

In one embodiment, a label is coupled to a molecule, such as an antibodythat specifically binds to SAP, through a chemical linker. Linkerdomains are typically polypeptide sequences, such as poly-gly sequencesof between about 5 and 200 amino acids. In some embodiments, prolineresidues are incorporated into the linker to prevent the formation ofsignificant secondary structural elements by the linker. Preferredlinkers are often flexible amino acid subsequences that are synthesizedas part of a recombinant fusion protein comprising the RNA recognitiondomain. In one embodiment, the flexible linker is an amino acidsubsequence that includes a proline, such as Gly(x)-Pro-Gly(x) (SEQ IDNO:5) where x is a number between about 3 and about 100. In otherembodiments, a chemical linker is used to connect synthetically orrecombinantly produced recognition and labeling domain subsequences.Such flexible linkers are known to persons of skill in the art. Forexample, poly(ethylene glycol) linkers are available from ShearwaterPolymers, Inc. Huntsville, Ala. These linkers optionally have amidelinkages, sulfhydryl linkages, or heterofunctional linkages.

The detectable labels used in the assays of the present invention, whichare attached to the detection reagent, can be primary labels (where thelabel comprises an element that is detected directly or that produces adirectly detectable element) or secondary labels (where the detectedlabel binds to a primary label, e.g., as is common in immunologicallabeling). An introduction to labels, labeling procedures and detectionof labels is found in Polak and Van Noorden (1997) Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, NY and in Haugland (1996)Handbook of Fluorescent Probes and Research Chemicals, a combinedhandbook and catalogue Published by Molecular Probes, Inc., Eugene,Oreg. Patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241.

Primary and secondary labels can include undetected elements as well asdetected elements. Useful primary and secondary labels in the presentinvention can include spectral labels such as green fluorescent protein,fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴c, ³²P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

Preferred labels include those that use: 1) chemiluminescence (usinghorseradish peroxidase and/or alkaline phosphatase with substrates thatproduce photons as breakdown products as described above) with kitsbeing available, e.g., from Molecular Probes, Amersham,Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) colorproduction (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored product (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) fluorescence using, e.g., an enzyme such as alkaline phosphatase,together with the substrate AttoPhos (Amersham) or other substrates thatproduce fluorescent products, 4) fluorescence (e.g., using Cy-5(Amersham), fluorescein, and other fluorescent tags); 5) radioactivity.Other methods for labeling and detection will be readily apparent to oneskilled in the art.

For use of the present invention outside the laboratory, preferredlabels are non-radioactive and readily detected without the necessity ofsophisticated instrumentation. Preferably, detection of the labels willyield a visible signal that is immediately discernable upon visualinspection. One preferred example of detectable secondary labelingstrategies uses an antibody that recognizes SAP in which the antibody islinked to an enzyme (typically by recombinant or covalent chemicalbonding). The antibody is detected when the enzyme reacts with itssubstrate, producing a detectable product. Preferred enzymes that can beconjugated to detection reagents of the invention include, e.g.,β-galactosidase, luciferase, horse radish peroxidase, and alkalinephosphatase. The chemiluminescent substrate for luciferase is luciferin.One embodiment of a fluorescent substrate for β-galactosidase is4-methylumbelliferyl-β-D-galactoside. Embodiments of alkalinephosphatase substrates include p-nitrophenyl phosphate (pNPP), which isdetected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′-amino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer;and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4ClN), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

The presence of a label can be detected by inspection, or a detectorwhich monitors a particular probe or probe combination can be used todetect the detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

B. B. anthracis Protein Surface Array Protein (SAP) Nucleic Acids andPolypeptides

The binding reagents used in the assays and kits of the invention aregenerally obtained using a B. anthracis SAP polypeptide as an immunogen.The entire SAP can be used, or polypeptide subfragments that include animmunogenic epitope can be used. Suitable SAP immunogens can be isolatedfrom B. anthracis cultures, or more preferably can be produced usingrecombinant methods.

1. SAP Polypeptides

SAP polypeptides can be produced by methods known to those of skill inthe art. The amino acid sequence of a B. anthracis SAP polypeptide isprovided as SEQ ID NO:1. A B. anthracis SAP polypeptide from a differentstrain is described in Etienne-Toumelin et al., J. Bacteriol.177:614-620 (1995).

In a presently preferred embodiment, the SAP proteins, or immunogenicsubsequences thereof, are synthesized using recombinant DNA methodology.Generally this involves creating a DNA sequence that encodes thepolypeptide, modified as desired, placing the DNA in an expressioncassette under the control of a particular promoter, expressing theprotein in a host, isolating the expressed protein and, if required,renaturing the protein.

SAP polypeptides can be expressed in a variety of host cells, includingE. coli, other bacterial hosts, yeasts, filamentous fungi, and varioushigher eukaryotic cells such as the COS, CHO and HeLa cells lines andmyeloma cell lines. Techniques for gene expression in microorganisms aredescribed in, for example, Smith, Gene Expression in RecombinantMicroorganisms (Bioprocess Technology, Vol. 22), Marcel Dekker, 1994.Examples of bacteria that are useful for expression include, but are notlimited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus. Filamentous fungi that areusefall as expression hosts include, for example, the following genera:Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium,Achlya, Podospora, Mucor, Cochliobolus, and Pyricularia. See, e.g., U.S.Pat. No. 5,679,543 and Stahl and Tudzynski, Eds., Molecular Biology inFilamentous Fungi, John Wiley & Sons, 1992. Synthesis of heterologousproteins in yeast is well known and described in the literature. Methodsin Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory,(1982) is a well recognized work describing the various methodsavailable to produce the enzymes in yeast.

SAP proteins, whether recombinantly or naturally produced, can bepurified, either partially or substantially to homogeneity, according tostandard procedures of the art, such as, for example, ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Once purified, partially or to homogeneity asdesired, the polypeptides can then be used (e.g., as immunogens forantibody production).

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the SAP protein(s) may possess aconformation substantially different than the native conformations ofthe constituent polypeptides. In this case, it may be necessary ordesirable to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem.268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem. 4:581-585; and Buchner et al. (1992) Anal. Biochem. 205: 263-270).Debinski et al., for example, describe the denaturation and reduction ofinclusion body proteins in guanidine-DTE. The protein is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

One of skill also would recognize that modifications can be made to theSAP polypeptides without diminishing their immunogenic activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the polypeptide into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

2. B. anthracis SAP-Encoding Nucleic Acids

Nucleic acids that encode B. anthracis are useful for the recombinantproduction of SAP. Such nucleic acids can be isolated, for example, byroutine cloning methods. The cDNA sequence provided in SEQ ID NO:2 canbe used to provide probes that specifically hybridize to a SAP gene, toa SAP mRNA, or to a SAP cDNA in a cDNA library (e.g., in a Southern orNorthern blot). Once the target SAP nucleic acid is identified, it canbe isolated according to standard methods known to those of skill in theart (see, e.g., Sambrook, Berger, and Ausubel, supra.).

SAP nucleic acids also can be isolated by amplification methods such aspolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR). A wide variety of cloning and invitro amplification methodologies are well-known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger,Sambrook, and Ausubel (all supra.); Cashion et al., U.S. Pat. No.5,017,478; and Carr, European Patent No. 0,246,864. Examples oftechniques sufficient to direct persons of skill through in vitroamplification methods are found in Berger, Sambrook, and Ausubel, aswell as Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols AGuide to Methods and Applications (Innis et al., eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al.(1989) Proc. Nat'l. Acad. Sci. USA 86: 1173; Guatelli et al. (1990)Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem.35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt(1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene, 4: 560; andBarringer et al. (1990) Gene 89: 117.

A polynucleotide that encodes a SAP polypeptide can be operably linkedto appropriate expression control sequences for a particular host cellin which the polypeptide is to be expressed. Such constructs are oftenreferred to as “expression cassettes.” For E. coli, appropriate controlsequences include a promoter such as the T7, trp, or lambda promoters, aribosome binding site and preferably a transcription termination signal.For eukaryotic cells, the control sequences typically include a promoterwhich optionally includes an enhancer derived from immunoglobulin genes,SV40, cytomegalovirus, etc., and a polyadenylation sequence, and mayinclude splice donor and acceptor sequences. In yeast, convenientpromoters include GAL1,10 (Johnson and Davies (1984) Mol. Cell. Biol.4:1440-1448) ADH2 (Russell et al. (1983) J. Biol. Chem. 258:2674-2682),PHO5 (EMBO J. (1982) 6:675-680), and MFα1 (Herskowitz and Oshima (1982)in The Molecular Biology of the Yeast Saccharomyces (eds. Strathem,Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.,pp. 181-209).

Expression cassettes are typically introduced into a vector thatfacilitates entry into a host cell, and maintenance of the expressioncassette in the host cell. Vectors that include a polynucleotide thatencodes a SAP polypeptide are provided by the invention. Such vectorsoften include an expression cassette that can drive expression of theSAP polypeptide. To easily obtain a vector of the invention, one canclone a polynucleotide that encodes the SAP polypeptide into acommercially or commonly available vector. A variety of common vectorssuitable for this purpose are well known in the art. For cloning inbacteria, common vectors include pBR322 derived vectors such aspBLUESCRIP™, and α-phage derived vectors. In yeast, vectors includeYeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids(the YRp series plasmids) and pGPD-2. A multicopy plasmid with selectivemarkers such as Leu-2, URA-3, Trp-1, and His-3 is also commonly used. Anumber of yeast expression plasmids such as YEp6, YEp 13, YEp4 can beused as expression vectors. The above-mentioned plasmids have been fullydescribed in the literature (Botstein et al. (1979) Gene 8:17-24; Broachet al. (1979) Gene, 8:121-133). For a discussion of yeast expressionplasmids, see, e.g., Parents, B., YEAST (1985), and Ausubel, Sambrook,and Berger, all supra). Expression in mammalian cells can be achievedusing a variety of commonly available plasmids, including pSV2, pBC12BI,and p91023, as well as lytic virus vectors (e.g., vaccinia virus,adenovirus, and baculovirus), episomal virus vectors (e.g., bovinepapillomavirus), and retroviral vectors (e.g., murine retroviruses).

The nucleic acids that encode SAP polypeptides can be transferred intothe chosen host cell by well-known methods such as calcium chloridetransformation for E. coli and calcium phosphate treatment orelectroporation for E. coli or mammalian cells. Cells transformed by theplasmids can be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, gpt, neo and hyg genes,among others. Techniques for transforming fungi are well known in theliterature and have been described, for instance, by Beggs et al.((1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933), Yelton et al. ((1984)Proc. Natl. Acad. Sci. USA 81: 1740-1747), and Russell ((1983) Nature301: 167-169). Procedures for transforming yeast are also well known(see, e.g., Beggs (1978) Nature (London), 275:104-109; and Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA, 75:1929-1933. Transformation andinfection methods for mammalian and other cells are described in Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook etal.); Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1994 Supplement)(Ausubel).

C. Assay Formats

The B. anthracis detection methods of the present invention can becarried out in a wide variety of assay formats. Typically, the assaymethods involve immobilization of B. anthracis SAP on a solid support,followed by detection of the immobilized SAP. The detectable labels canbe detected directly after immobilization on the solid support, forexample, or indirectly by an enzymatic or other reaction that results ina detectable change in a reactant that is present in the detection assayreaction.

1. ELISA Detection Methods of the Invention

Presently preferred assay systems for use in the kit and methods of theinvention are based on the enzyme-linked immunosorbent assay (ELISA)method. General methods for ELISA are well known to those of skill inthe art (see, e.g., Elder et al., J. Clin. Microbiol. 16:141 (1982);Ausubel et al., supra). Generally, antigens fixed to a solid surface aredetected using antigen-specific antibodies that are detected by way ofan enzymatic reaction. In a presently preferred embodiment, the ELISAmethod used is the “sandwich” method wherein the antigens are bound tothe solid surface via an antibody bound to the solid surface. A secondantibody, typically linked to an enzyme, is then contacted to theantigen, washed, then contacted with the enzyme substrate to selectbinding. These and other embodiments of the ELISA method are taught in,for example, Ausubel et al. § 11.2, supra.

To immobilize SAP on the solid support, a capture reagent thatspecifically binds to SAP is non-diffusively associated with thesupport. The capture reagents can be non-diffusively immobilized on thesupport either by covalent or non-covalent methods, which are known tothose of skill in the art. See, e.g., Pluskal et al. (1986)BioTechniques 4: 272-283. Suitable supports include, for example,glasses, plastics, polymers, metals, metalloids, ceramics, organics, andthe like. Specific examples include, but are not limited to, microtiterplates, nitrocellulose membranes, nylon membranes, and derivatized nylonmembranes, beads, and also particles, such as agarose, SEPHADEX™, andthe like. Assay systems for use in the methods and kits of the inventioninclude, but are not limited to, dipstick-type devices,immunochromatographic test strips and radial partition immunoassaydevices, microtiter assays and flow-through devices. Conveniently, wherethe solid support is a membrane, the test sample can flow through themembrane, for example, by gravity, capillary action, or under positiveor negative pressure.

Once the test sample has been contacted with the solid support, thesolid support is then contacted with detection reagents for SAP. Thesolid support can be washed prior to contact with detection reagents toremove unbound reagents and test sample components. After incubation ofthe detection reagents for a sufficient time to bind a substantialportion of the immobilized SAP, any unbound labeled reagents are removedby, for example, washing. The detectable label associated with thedetection reagents is then detected. For example, in the case of anenzyme used as a detectable label, a substrate for the enzyme that turnsa visible color upon action of the enzyme is placed in contact with thebound detection reagent. A visible color will then be observed inproportion to the amount of the specific antigen in the sample.

2. Membrane-Based Detection Methods of the Invention

In some embodiments, the assay methods are carried out using amembrane-based detection system such as are described in U.S. Pat. No.5,922,615 and EP 447154. These systems employ an apparatus that includesa porous member, such as a membrane or a filter, onto which is bound amultiplicity of capture reagents that specifically bind B. anthracisSAP. The apparatus also includes a non-absorbent member with a texturedsurface in communication with the lower surface of the porous member.The textured surface of the non-absorbent member can be a groovedsurface (e.g., analogous to the surface of a record album) or it can becomposed of channels, such that when the porous and non-absorbentmembers are brought into contact with one another a network of capillarychannels is formed. The capillary network is formed from the contact ofthe porous member with the textured surface of the non-absorbent memberand can be constructed either before or subsequent to the initialcontacting of the porous member with a fluid.

In some embodiments, the capillary communication between the porousmember and the non-absorbent member favors delaying the transferal offluid from the porous member to the capillary network formed by theporous member and the textured surface of the non-absorbent member untilthe volume of the added fluid substantially exceeds the void volume ofthe porous member. The transferal of fluid from the porous member to thenetwork of capillary channels formed by the porous member and thetextured surface of the non-absorbent member can occur without the useof external means, such as positive external pressure or vacuum, orcontact with an absorbent material.

The devices of the present invention can also include an optional memberwhich is placed in contact with the upper surface of the porous memberand may be used to partition the upper surface of the device intodiscrete openings. Such openings can access either the porous member orthe textured surface of the non-absorbent second member. The optionalmember can in conjunction with the non-absorbent member compose a fluidreceiving zone in which there is no intervening porous member. A fluidreceiving zone constructed from the non-absorbent member and theoptional member provides fluid capacity in addition to that provided bythe network of capillary channels created by the contact of the porousmember and the non-absorbent member. The openings in the optional membermay include a first fluid opening and also an additional fluid opening.The first fluid opening functions as a portal for the introduction ofthe first fluid added to the device. The additional fluid opening servesas an additional portal through which additional fluids may be added tothe inventive device.

To perform an assay using these devices, a volume of the test sample isadded to the porous member, where the sample permeates the void volumeof the porous member and thereby contacts the anchor moietiesimmobilized on the porous member. In a non-competitive assay, the sampleto be assayed is applied to the porous member and the SAP, if present,is bound by the anchor moieties. A detection reagent for SAP is thenadded as an additional fluid; these bind to the complex of SAP andcapture reagent. Alternatively, the detection reagent can be added tothe sample prior to application of the sample to the porous member sothat the binding of detection reagent to SAP occurs prior to the bindingof SAP to the capture reagent. In another embodiment, the capturereagent and detection reagent are added to the sample, after which thecomplex of capture reagent, SAP, and detection reagent binds to abinding agent that is either combined with these reagents or isimmobilized on the porous member. An additional fluid containingreagents to effect a separation of free from bound labeled reagents canbe added to remove excess detection reagent, if needed.

This device is designed to provide sufficient sensitivity to measure lowconcentrations of SAP because one can use large amounts of sample andefficiently remove the excess of detection reagent. Indeed, theefficient separation of free from bound label achieved by the network ofcapillary channels of this device improves the discrimination ofspecific SAP-associated signal over non-specific background signal. Ifneeded, a signal developer solution is then added to enable the label ofthe detection moiety to develop a detectable signal. The signaldeveloped can then be related to the concentration of the target ligandwithin the sample. In a preferred embodiment, the transfer of fluidbetween the porous first member of the device and the network ofcapillary channels formed by the contact of the porous member andtextured surface of the non-absorbent second member of the device isgenerally self-initiated at the point when the total volume of fluidadded to the device exceeds the void volume of the porous member, thusobviating the need for active interaction by the user to remove excessfluid from the analyte detection zone. The point at which the fluidtransfer is initiated is dependent upon the objectives of the assay.Normally, it is desirable to contact the sample with all of the zones onthe porous member which contain immobilized receptor. This methodenables the detection of SAP in a manner that is simple, rapid,convenient, sensitive and efficient in the use of reagents.

Competitive binding assays can also be used to detect SAP. Conveniently,these assays are performed using the described devices by adding to asample a labeled analog of SAP. The labeled analog and SAP present inthe sample compete for the binding sites of the capture reagents.Alternatively, the capture reagents can be combined with the sample andlabeled analogs with subsequent immobilization of the capture reagentsonto the porous member through contact with a binding agent. Anadditional fluid to separate the free from bound label may be added tothe device, followed if needed by a signal development solution toenable detection of the label of the labeled analog which has complexedwith capture reagent immobilized on the porous member. The amount oflabeled SAP bound to the porous member is related to the concentrationof SAP in the sample.

D. Kits for Detecting Anthrax

This invention also provides kits for the detection and/orquantification of anthrax using the methods described herein. The kitscan include a container containing one or more of the above-discusseddetection reagents with or without labels, and capture reagents, eitherfree or bound to solid supports. A suitable solid support, such as amembrane, can also be included in the kits of the invention. The kitscan provide the solid supports in the form of an assay apparatus that isadapted to use in the described assay. Preferably, the kits will alsoinclude reagents used in the described assays, including reagents usefulfor detecting the presence of the detectable labels. Other materialsuseful in the performance of the assays can also be included in thekits, including test tubes, transfer pipettes, and the like. The kitscan also include written instructions for the use of one or more ofthese reagents in any of the assays described herein.

The kits of the invention can also include an internal and/or anexternal control. An internal control can consist of the SAPpolypeptide. The control antigen can conveniently be preattached to acapture reagent in a zone of the solid support adjacent to the zone towhich the sample is applied. The external control can also consist ofthe SAP polypeptide. Typically, the antigen present in the externalcontrol will be at a concentration at or above the sensitivity limit ofthe assay means. The external control antigen can be diluted in thesample diluent and assayed in the same manner as would a biologicalsample. Alternatively, the external control SAP polypeptide can be addedto an aliquot of an actual biological sample to determine thesensitivity of the assay. The kits of the present invention can containmaterials sufficient for one assay, or can contain sufficient materialsfor multiple assays.

E. Test Samples

Samples to test for the presence of anthrax can be collected from anypotential source of anthrax. Samples can be collected from, for example,the air, water, food, soil or other solids or liquids. In oneembodiment, the methods and kits of the invention can be used todetermine if terrorists have planted anthrax in a public area. Inpreferred embodiments, it is unknown whether the test sample contains B.anthracis.

Air samples can be collected using, for example, a cyclonic collectiondevice (see, e.g., Jensen et al., Am. Ind. Hyg. Assoc. J. 53:660-67(1992); Cage et al., Ann. Allergy Asthma Immunol. 77:401-6 (1996)). Sucha device can capture a volume of air, submit the air to turbulence suchthat any particles in the air (e.g., anthrax spores or SAP-coatedparticles) are deposited on a moist surface. Typically, air flowingthrough cyclonic tubes forms a vortex in the tube that induces highcentrifugal forces on particles in the air (Anderson et al., JohnsHopkins APL Technical Digest 20(3) (1999)). The rotational forcessegregate the larger particle to the outside of the tube. Variations inthe tube diameter, length, taper angle and flow velocity determineparticle separation size. Particles can then be captured by letting theparticles slide down the tube walls into a filter bag or by washing thewalls with a liquid and capturing the concentrate. The objects can thenbe collected and analyzed for the presence of anthrax. A variety ofcyclonic devices are discussed in, e.g., Maddox et al. MonthlyMicroscopical J. 286-290 (1870); Fisher-Klosterman, Inc. ProductBulletin 218-C, 2900 West Broadway, Louisville, Ky.; Hering, “Impactors,Cyclones, and Other Inertial and Gravitational Collectors,” in AirSampling Instruments for Evaluation of Atmospheric contaminants, 8thEd., American Conference of Governmental Industrial Hygienists,Cincinnati, Ohio, 279-321 (1995) and; Stoutas, et al. J. Aerosol Sci.25(7):1321-1330 (1994). Handheld air samplers can also be used to obtainsamples that are tested according to the methods of the invention (see,e.g., Kenny et al., Am. Ind. Hyg. Assoc. J. 59:831-41 (1998)). Samplingof solid or liquid objects is known to those skilled in the art.

Several cyclonic collection devices are known, including conventionalimpactors and virtual impactors. Conventional impactors work bydirecting the particle-Containing air through a nozzle onto a collectionplate. A variation of the conventional impactor is the virtual impactor,which operates by directing the air stream from the nozzle to an openingwith a restricted flow. Larger particles enter an opening which forms avirtual surface, and become entrained in a minor flow or reducedvelocity, while smaller particles follow the major flow. The virtualimpactor has the benefit of concentrating particle quantity from lowdensity in the high volume flow to high density in the low volume flow.See, e.g., Anderson et al., supra.

A significant advantage of the assay methods and kits of the inventionis that the sensitivity is such that a sample need not be cultured priorto assay. This not only provides a faster and less expensive assay, butalso makes it possible to obtain a result in the field. Samples need notbe sent to a laboratory facility for processing. This is particularlyadvantageous in military situations, in which suitable laboratoryfacilities may not be available.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Isolation of a Gene Encoding Bacillus anthracis Surface ArrayProtein (SAP)

This Example describes the cloning and characterization of a gene thatencodes a Bacillus anthracis surface array protein (SAP).

Isolation of B. anthracis DNA

Bacillus anthracis genomic DNA isolated from the non-pathologic Sternestrain was used as a template source for PCR amplification of a nucleicacid that codes for SAP. A total of 6 ml of Bacillus anthracis Sternestrain (1×10¹⁰/ml in PBS pH 7.4) was pelleted in a microcentrifuge at10,000 g for 5 minutes. Bacterial pellets were then combined andresuspended in a final volume of 1 ml lysis buffer (50 mMTris(hydroxymethyl) aminomethane (“Tris”) pH 7.8, 10 mMethylenediaminetetraacetic acid (“EDTA”), 100 μg/ml Ribonuclease (RNaseA) (Roche Molecular Biochemical, Indianapolis, Ind.), 0.5% Triton X-100™(T-octylphenoxypolyethoxyethanol) (Sigma, St. Louis, Mo.), 12.5%sucrose). Lysozyme (Sigma) was added to a final concentration of 2 mg/mland the mixture incubated for 1 hr at 37° C. 300 μg of Proteinase K(Roche Molecular Biochemical, Indianapolis, Ind.) and a one-tenth volumeof 10% SDS was added to the mixture followed by a 1 hr incubation at 56°C. NaCl was then added to a final concentration of 500 mM by addingone-tenth volume of 5 M NaCl. The mixture was then twice extracted withphenol/chloroform (phenol:chloroform:isoamyl alcohol (50:49:1)) and theDNA in the aqueous layer sheared by passing the solution through an 18gauge needle. DNA was precipitated with 2.5 volumes of ethanol andresuspended in 200 μl of distilled water. This DNA preparation wasextracted once with Tris pH 8 equilibrated phenol, two times withphenol/chloroform and finally twice with chloroform (chloroform:isoamylalcohol (49:1)) alone. DNA in the aqueous layer was precipitated a finaltime and resuspended in 500 μl of distilled water, yieldingapproximately 79 μg of DNA at 158 μg/ml. This DNA was used as a templatein the subsequent PCR amplification of the SAP gene.

Cloning of Bacillus anthracis SAP Gene via PCR

Appropriate PCR primers were made corresponding to the coding sequenceof the 5′ and 3′ ends of the B. anthracis SAP gene (see primer sequencebelow). These primers were based on a published nucleotide sequence(Etienne-Toumelin et al, supra). DNA encoding the native signal sequenceof SAP (amino acids 1-29) was purposefully omitted from the cloningsince a functional signal sequence was provided by the expression vectorpBRncoH3 (described in copending, commonly-owned U.S. patent applicationSer. No. 08/835,159, filed Apr. 4, 1997). The 5′ primer contains 23bases of vector sequence at its 5′-end that corresponds to the 3′-end ofthe pBRncoH3 vector. The 3′ primer contains 19 bases of the tetracyclinepromoter, removed by HindIII digestion in the vector, in addition to 20bases of vector sequence 3′ to the HindIII site. The 3′ primer was alsoengineered to encode a hexahistidine amino acid tag at the C-terminus ofthe SAP protein to allow for efficient purification using nickel-chelateaffinity chromatography (see below).

-   5′ PCR primer: 5′-TCGCTGCCCAACCAGCCATGGCCGCAGGTAAAA CATTCCCAGAC-3′    (SEQ ID NO:3)-   3′ PCR primer: 5′-GTGATAAACTACCGCATTAAAGCTTATCGATGATA    AGCTGTCAATTAGTGATGGTGATGGTGATGTTTTG TTGCAGGTTTTGCTTCTTT-3′ (SEQ ID    NO:4)

The nucleic acid that encodes SAP was amplified using these primers andapproximately 30 ng of Bacillus anthracis genomic DNA as template. Theamplification was performed using Expand™ DNA polymerase (RocheMolecular Biochemical (Indianapolis, Ind.). SAP insert DNA (˜300 ng) waspurified and annealed to the HindIII-digested pBRncoH3 vector (100 ng)at a 6:1 molar ratio of insert to vector. An aliquot was electroporatedinto 40 μl of electrocompetent E. coli strain DH10B as described inExample 3. Various dilutions of the transformed cells were plated on LBagar plates supplemented with tetracycline (10 μg/ml) and grownovernight at 37° C. Three colonies were each picked into 3 ml 2xYT,supplemented with tetracycline (10 μg/ml), and grown overnight at 37° C.The following day, glycerol freezer stocks were made for long termstorage at −80° C.

In order to confirm that the SAP gene had indeed been cloned, each ofthe three clones was tested for the ability to synthesize SAP proteinupon arabinose induction as described below. All three clones produced aprotein of the predicted size, approximately 94 kDa in molecular mass,and were shown to react with a rabbit anti-anthracis polyclonal serumusing Western blot analysis (data not shown). Two of the three cloneswere sequenced and compared against the National Center forBiotechnology Information's (NCBI) non-redundant nucleotide databaseusing the BLAST search engine. This search indicated that a SAP gene hadindeed been cloned. There were eight differences in the predicted aminoacid sequence compared to the noted-published sequence. These changesare lysine 264 to arginine, glutamic acid 478 to alanine, arginine 482to histidine, glutamic acid 496 to aspartic acid, lysine 556 toarginine, glutamic acid 606 to aspartic acid, lysine 607 to threonine,and valine 751 to alanine. Amino acid numbering is based on thepublished sequence (Etienne-Toumelin et al., supra). These differencesmay be due to the fact that a different Bacillus anthracis strain wasused in the work described here. The original published work did not usethe Sterne strain. The predicted amino acid sequence of the SAP genecloned here shows 8 amino acid differences out of 785, and is thus 99.0%identical to the published sequence.

Example 2 Expression and Purification of Recombinant Bacllus anthracisSap from E. Coli

This Example describes the expression and purification of B. anthracisSAP using E. coli.

A shake flask containing 2xYT supplemented with 1% glycerol wasinoculated with an E. coli DH10B strain from Example 1 that contained acloned B. anthracis SAP gene and incubated overnight in an Innova 4330incubator shaker (New Brunswick Scientific, Edison, N.J.) set at 37° C.,300 rpm. The inoculum was used to seed 500 mL cultures of defined medium(Pack et al. (1993) Bio/Technology 11: 1271-1277) supplemented with 3g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco, Detroit,Mich.), 12.5 g/L glycerol and 10 μg/ml tetracycline. Cultures were grownin 2 L Tunair shake flasks (Shelton Scientific, Shelton, Conn.) at 37°C. and 300 rpm. Cells were grown to an optical density of approximately4 absorption units at 600 nm. Expression of SAP was then induced byaddition of L(+)-arabinose to 2 g/L during this logarithmic growthphase. The flasks were then maintained at 23° C. and 300 rpm overnight.

The following morning, bacterial cultures were passed through an M-110YMicrofluidizer (Microfluidics, Newton, Mass.) at 17,000 psi. Thehomogenate was clarified in a J2-21 centrifuge (Beckman, Fullerton,Calif.) and recombinant SAP purified from the supernatant usingimmobilized metal affinity chromatography. Briefly, Chelating SepharoseFastFlow™ resin (Pharmacia, Piscataway, N.J.) was charged with 0.1 MNiCl₂ and equilibrated in 20 mM borate, 150 mM NaCl, 10 mM imidazole,0.01% NaN₃, pH 8. A stock solution was used to bring the supernatantconcentration to 10 mM imidazole, pH 8. Chelating resin was then addedto the supernatant and the mixture shaken for 1 hour at roomtemperature, 150-200 rpm. During this time, SAP was captured by means ofthe high affinity interaction between nickel and the hexahistidine tagengineered onto the C-terminus of SAP. After 1 hour, the resin mixturewas poured into a chromatography column and washed with 20 mM borate,150 mM NaCl, 10 mM imidazole, 0.01% NaN₃, pH 8.0. SAP was eluted fromthe resin with the same buffer containing 200 mM imidazole instead of 10mM.

The volume of eluted SAP was reduced using a centrifuge concentratorwith a 30 kDa molecular weight cut off (Amicon, Beverly, Mass.), and thesample subsequently dialyzed against sterile phosphate-buffered solution(PBS) for immunizations and BBS (20 mM borate, 150 mM NaCl, 0.01% NaN₃,pH 8.0) for biotinylation. Isolated SAP was evaluated for purity bySDS-PAGE analysis and shown to be greater than 95% pure. The proteinconcentration of recombinant SAP was determined by UV absorbance at 280nm, assuming an absorbance of 0.593 for a 1 mg/ml solution.

Example 3 Construction of a Phage Display Library

This Example describes the construction of a phage display library fromwhich binding reagents that are specific for B. anthracis SAP wereidentified.

Immunization and mRNA Isolation

A phage display library for identification of SAP-binding molecules wasconstructed as follows. A/J mice (Jackson Laboratories, Bar Harbor, Me.)were immunized intraperitoneally with recombinant SAP antigen, using 100μg protein in Freund's complete adjuvant, on day 0, and with 100 μgantigen on day 28. Test bleeds of mice were obtained through puncture ofthe retro-orbital sinus. If, by testing the titers, they were deemedhigh by ELISA using biotinylated SAP antigen immobilized via neutravidin(Reacti-Bind™ NeutrAvidin™-Coated Polystyrene Plates, Pierce, Rockford,Ill.), the mice were boosted with 100 μg of protein on day 70, 71 and72, with subsequent sacrifice and splenectomy on day 77. If titers ofantibody were not deemed satisfactory, mice were boosted with 100 μgantigen on day 56 and a test bleed taken on day 63. If satisfactorytiters were obtained, the animals were boosted with 100 μg of antigen onday 98, 99, and 100 and the spleens harvested on day 105.

The spleens were harvested in a laminar flow hood and transferred to apetri dish, trimming off and discarding fat and connective tissue. Thespleens were macerated quickly with the plunger from a sterile 5 ccsyringe in the presence of 1.0 ml of solution D (25.0 g guanidinethiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3 ml sterilewater, 1.76 ml 0.75 M sodium citrate pH 7.0, 2.64 ml 10% sarkosyl(Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercantoethanol (FisberScientific. Pittsburgh, Pa.)). This spleen suspension was pulled throughan 18 gauge needle until all cells were lysed and the viscous solutionwas transferred to a microcentrifuge tube. The petri dish was washedwith 100 μl of solution D to recover any remaining spleen. Thissuspension was then pulled through a 22 gauge needle an additional 5-10times.

The sample was divided evenly between two microcentrifuge tubes and thefollowing added, in order, with mixing by inversion after each addition:50 μl 2 M sodium acetate pH 4.0, 0.5 ml water-saturated phenol (FisherScientific, Pittsburgh, Pa.), 100 μl chloroform/isoamyl alcohol 49:1(Fisher Scientific, Pittsburgh, Pa.). The solution was vortexed for 10seconds and incubated on ice for 15 min. Following centrifugation at 14krpm for 20 min at 2-8° C., the aqueous phase was transferred to a freshtube. An equal volume of water saturated phenol:chloroform:isoamylalcohol (50:49:1) was added, and the tube vortexed for ten seconds.After a 15 min incubation on ice, the sample was centrifuged for 20 minat 2-8° C., and the aqueous phase transferred to a fresh tube andprecipitated with an equal volume of isopropanol at −20° C. for aminimum of 30 min. Following centrifugation at 14 krpm for 20 min at 4°C., the supernatant was aspirated away, the tubes briefly spun and alltraces of liquid removed from the RNA pellet.

The RNA pellets were each dissolved in 300 μl of solution D, combined,and precipitated with an equal volume of isopropanol at −20° C. for aminimum of 30 min. The sample was centrifuged 14 krpm for 20 min at 4°C., the supernatant aspirated as before, and the sample rinsed with 100μl of ice-cold 70% ethanol. The sample was again centrifuged 14 krpm for20 min at 4° C., the 70% ethanol solution aspirated, and the RNA pelletdried in vacuo. The pellet was resuspended in 100 μl of sterile diethylpyrocarbonate-treated water. The concentration was determined by A₂₆₀using an absorbance of 1.0 for a concentration of 40 μg/ml. The RNAswere stored at −80° C.

Preparation of Complementary DNA (cDNA)

The total RNA purified from mouse spleens as described above was useddirectly as template for cDNA preparation. RNA (50 μg) was diluted to100 μL with sterile water, and 10 μL of 130 ng/μL oligo dT₁₂(synthesized on Applied Biosystems Model 392 DNA synthesizer) was added.The sample was heated for 10 min at 70° C., then cooled on ice. Forty μL5× first strand buffer was added (Gibco/BRL, Gaithersburg, Md.), alongwith 20 μL 0.1 M dithiothreitol (Gibco/BRL, Gaithersburg, Md.), 10 μL 20mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim,Indianapolis, Ind.), and 10 μL water on ice. The sample was thenincubated at 37° C. for 2 min. Ten μL reverse transcriptase(Superscript™ II, Gibco/BRL, Gaithersburg, Md.) was added and incubationwas continued at 37° C. for 1 hr. The cDNA products were used directlyfor polymerase chain reaction (PCR).

Amplification of Antibody Genes by PCR

To amplify substantially all of the H and L chain genes using PCR,primers were chosen that corresponded to substantially all publishedsequences. Because the nucleotide sequences of the amino termini of Hand L contain considerable diversity, 33 oligonucleotides weresynthesized to serve as 5′ primers for the H chains, and 29oligonucleotides were synthesized to serve as 5′ primers for the kappa Lchains as described in U.S. patent application Ser. No. 08/835,159,filed Apr. 4, 1997. The constant region nucleotide sequences for eachchain required only one 3′ primer for the H chains and one 3′ primer forthe kappa L chains.

A 50 μL reaction was performed for each primer pair with 50 pmol of 5′primer, 50 pmol of 3′ primer, 0.25 μL Taq DNA Polymerase (5 units/μL,Boehringer Mannheim, Indianapolis, Ind.), 3 μL cDNA (prepared asdescribed in Example 3), 5 μL 2 mM dNTP's, 5 μL 10× Taq DNA polymerasebuffer with MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to50 μL. Amplification was done using a GeneAmp® 9600 thermal cycler(Perkin Elmer, Foster City, Calif.) with the following thermocycleprogram: 94° C. for 1 min; 30 cycles of 94° C. for 20 sec, 55° C. for 30sec, and 72° C. for 30 sec; 72° C. for 6 min; 4° C.

The dsDNA products of the PCR process were then subjected to asymmetricPCR using only a 3′ primer to generate substantially only the anti-sensestrand of the target genes. A 100 μL reaction was done for each dsDNAproduct with 200 pmol of 3′ primer, 2 μL of ds-DNA product, 0.5 μL TaqDNA Polymerase, 10 μL 2 mM dNTP's, 10 μL 10× Taq DNA polymerase bufferwith MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to 100 μL.The same PCR program as that described above was used to amplify thesingle-strainded (ss)-DNA.

Purification of Single-Stranded DNA by High Performance LiquidChromatography and Kinasing Single-Stranded DNA

The H chain ss-PCR products and the L chain single-stranded PCR productswere ethanol precipitated by adding 2.5 volumes ethanol and 0.2 volumes7.5 M ammonium acetate and incubating at −20° C. for at least 30 min.The DNA was pelleted by centrifuging in an Eppendorf centrifuge at 14krpm for 10 min at 2-8° C. The supernatant was carefully aspirated, andthe tubes were briefly spun a 2nd time. The last drop of supernatant wasremoved with a pipette. The DNA was dried in vacuo for 10 min on mediumheat. The H chain products were pooled in 210 μL water and the L chainproducts were pooled separately in 210 μL water. The single-stranded DNAwas purified by high performance liquid chromatography (HPLC) using aHewlett Packard 1090 HPLC and a Gen-Pak™ FAX anion exchange column(Millipore Corp., Milford, Mass.). The gradient used to purify thesingle-stranded DNA is shown in Table 1, and the oven temperature was60° C. Absorbance was monitored at 260 nm. The single-stranded DNAeluted from the HPLC was collected in 0.5 min fractions. Fractionscontaining single-stranded DNA were ethanol precipitated, pelleted anddried as described above. The dried DNA pellets were pooled in 200 μLsterile water.

TABLE 1 HPLC gradient for purification of ss-DNA Time (min) % A % B % CFlow (ml/min) 0 70 30 0 0.75 2 40 60 0 0.75 17 15 85 0 0.75 18 0 100 00.75 23 0 100 0 0.75 24 0 0 100 0.75 28 0 0 100 0.75 29 0 100 0 0.75 340 100 0 0.75 35 70 30 0 0.75 Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0Buffer B is 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 8.0 Buffer C is 40 mmphosphoric acid

The single-stranded DNA was 5′-phosphorylated in preparation formutagenesis. Twenty-four μL 10× kinase buffer (United StatesBiochemical, Cleveland, Ohio), 10.4 μL 10 mM adenosine-5′-triphosphate(Boehringer Mannheim, Indianapolis, Ind.), and 2 μL polynucleotidekinase (30 units/μL, United States Biochemical, Cleveland, Ohio) wasadded to each sample, and the tubes were incubated at 37° C. for 1 hr.The reactions were stopped by incubating the tubes at 70° C. for 10 min.The DNA was purified with one extraction of Tris equilibrated phenol(pH>8.0, United States Biochemical, Cleveland, Ohio):chloroform:isoamylalcohol (50:49:1) and one extraction with chloroform:isoamyl:alcohol(49:1). After the extractions, the DNA was ethanol precipitated andpelleted as described above. The DNA pellets were dried, then dissolvedin 50 μL sterile water. The concentration was determined by measuringthe absorbance of an aliquot of the DNA at 260 nm using 33 μg/ml for anabsorbance of 1.0. Samples were stored at −20° C.

Preparation of Uracil Templates Used in Generation of Spleen AntibodyPhage Libraries

One ml of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight culture wasadded to 50 ml 2xYT in a 250 ml baffled shake flask. The culture wasgrown at 37° C. to OD₆₀₀=0.6, inoculated with 10 μl of a 1/100 dilutionof BS45 vector phage stock (described in U.S. patent application Ser.No. 08/835,159, filed Apr. 4, 1997) and growth continued for 6 hr.Approximately 40 ml of the culture was centrifuged at 12 krpm for 15minutes at 4° C. The supernatant (30 ml) was transferred to a freshcentrifuge tube and incubated at room temperature for 15 minutes afterthe addition of 15 μl of 10 mg/ml RNaseA (Boehringer Mannheim,Indianapolis, Ind.). The phage were precipitated by the addition of 7.5ml of 20% polyethylene glycol 8000 (Fisher Scientific, Pittsburgh,Pa.)/3.5M ammonium acetate (Sigma Chemical Co., St. Louis, Mo.) andincubation on ice for 30 min. The sample was centrifuged at 12 krpm for15 min at 2-8° C. The supernatant was carefully discarded, and the tubebriefly spun to remove all traces of supernatant. The pellet wasresuspended in 400 μl of high salt buffer (300 mM NaCl, 100 mM Tris pH8.0, 1 mM EDTA), and transferred to a 1.5 ml tube.

The phage stock was extracted repeatedly with an equal volume ofequilibrated phenol:chloroform:isoamyl alcohol (50:49:1) until no traceof a white interface was visible, and then extracted with an equalvolume of chloroform:isoamyl alcohol (49:1). The DNA was precipitatedwith 2.5 volumes of ethanol and ⅕ volume 7.5 M ammonium acetate andincubated 30 min at −20° C. The DNA was centrifuged at 14 krpm for 10min at 4° C., the pellet washed once with cold 70% ethanol, and dried invacuo. The uracil template DNA was dissolved in 30 μl sterile water andthe concentration determined by A₂₆₀ using an absorbance of 1.0 for aconcentration of 40 μg/ml. The template was diluted to 250 ng/mL withsterile water, aliquoted, and stored at −20° C.

Mutagenesis of Uracil Template with ss-DNA and Electroporation into E.Coli to Generate Antibody Phage Libraries

Antibody phage display libraries were generated by simultaneouslyintroducing single-stranded heavy and light chain genes onto a phagedisplay vector uracil template. A typical mutagenesis was performed on a2 μg scale by mixing the following in a 0.2 ml PCR reaction tube: 8 μlof (250 ng/μL) uracil template, 8 μL of 10× annealing buffer (200 mMTris pH 7.0, 20 mM MgCl₂, 500 mM NaCl), 3.33 μl of kinasedsingle-stranded heavy chain insert (100 ng/μL), 3.1 μl of kinasedsingle-stranded light chain insert (100 ng/μL), and sterile water to 80μl. DNA was annealed in a GeneAmp® 9600 thermal cycler using thefollowing thermal profile: 20 sec at 94° C., 85° C. for 60 sec, 85° C.to 55° C. ramp over 30 min, hold at 55° C. for 15 min. The DNA wastransferred to ice after the program finished. The extension/ligationwas carried out by adding 811 of 10× synthesis buffer (5 mM each dNTP,10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl₂, 20 mM DTT), 8 μL T4 DNAligase (1U/μL, Boehringer Mannheim, Indianapolis, Ind.), 8 μL diluted T7DNA polymerase (1U/μL, New England BioLabs, Beverly, Mass.) andincubating at 37° C. for 30 min. The reaction was stopped with 300 μL ofmutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA). The mutagenesisDNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μL of sterile water.

One microliter of mutagenesis DNA (500 ng) was transferred into 40 μlelectrocompetent E. coli DH12S (Gibco/BRL, Gaithersburg, Md.) usingelectroporation. The transformed cells were mixed with approximately 1.0ml of overnight XL-1 cells which were diluted with 2xYT broth to 60% theoriginal volume. This mixture was then transferred to a 15-ml sterileculture tube and 9 ml of top agar added for plating on a 150-mm LB agarplate. Plates were incubated for 4 hrs at 37° C. and then transferred to20° C. overnight. First round antibody phage were made by eluting phageoff these plates in 10 ml of 2xYT, spinning out debris, and taking thesupernatant. These samples are the antibody phage display libraries usedfor selecting antibodies against SAP. Efficiency of the electroporationswas measured by plating 10 μl of a 10⁻⁴ dilution of suspended cells onLB agar plates, follow by overnight incubation of plates at 37° C. Theefficiency was calculated by multiplying the number of plaques on the10⁴ dilution plate by 10⁶. Library electroporation efficiencies aretypically greater than 1×10⁷ phage under these conditions.

Transformation of E. coli by Electroporation

Electrocompetent E. coli cells were thawed on ice. DNA was mixed with 40μL of these cells by gently pipetting the cells up and down 2-3 times,being careful not to introduce an air bubble. The cells were transferredto a Gene Pulser cuvette (0.2 cm gap, BioRAD, Hercules, Calif.) that hadbeen cooled on ice, again being careful not to introduce an air bubblein the transfer. The cuvette was placed in the E. coli Pulser (BioRAD,Hercules, Calif.) and electroporated with the voltage set at 1.88 kVaccording to the manufacturer's recommendation. The transformed samplewas immediately resuspended in 1 ml of 2xYT broth or 1 ml of a mixtureof 400 μl 2xYT/600111 overnight XL-1 cells and processed as proceduresdictated.

Plating M13 Phage or Cells Transformed with Antibody Phage-DisplayVector Mutagenesis Reaction

Phage samples were added to 200 μL of an overnight culture of E. coliXL1-Blue when plating on 100 mm LB agar plates or to 600 μL of overnightcells when plating on 150 mm plates in sterile 15 ml culture tubes.After adding LB top agar (3 ml for 100 mm plates or 9 ml for 150 mmplates, top agar stored at 55° C. (see, Appendix A1, Sambrook et al.,supra.), the mixture was evenly distributed on an LB agar plate that hadbeen pre-warmed (37° C.-55° C.) to remove any excess moisture on theagar surface. The plates were cooled at room temperature until the topagar solidified. The plates were inverted and incubated at 37° C. asindicated.

Preparation of Biotinylated SAP and Biotinylated Antibodies

Concentrated recombinant SAP antigen (Example 2 above) was extensivelydialyzed into BBS (20 mM borate, 150 mM NaCl, 0.1% NaN₃, pH 8.0). Afterdialysis, 1 mg of SAP (1 mg/ml in BBS) was reacted with a 15 fold molarexcess of biotin-XX-NHS ester (Molecular Probes, Eugene, Oreg., stocksolution at 40 mM in DMSO). The reaction was incubated at roomtemperature for 90 min and then quenched with taurine (Sigma ChemicalCo., St. Louis, Mo.) at a final concentration of 20 mM. The biotinylatedreaction mixture was then dialyzed against BBS at 2-8° C. Afterdialysis, biotinylated SAP was diluted in panning buffer (40 mM Tris,150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH 7.5), aliquoted, and storedat −80° C. until needed.

Antibodies were reacted with 3-(N-maleimidylpropionyl)biocytin(Molecular Probes, Eugene, Oreg.) using a free cysteine located at thecarboxy terminus of the heavy chain. Antibodies were reduced by addingDTT to a final concentration of 1 mM for 30 min at room temperature.Reduced antibody was passed through a Sephadex G50 desalting columnequilibrated in 50 mM potassium phosphate, 10 mM boric acid, 150 mMNaCl, pH 7.0. 3-(N-maleimidylpropionyl)-biocytin was added to a finalconcentration of 1 mM and the reaction allowed to proceed at roomtemperature for 60 min. Samples were then dialyzed extensively againstBBS and stored at 2-8° C.

Preparation of Avidin Magnetic Latex

The magnetic latex (Estapor, 10% solids, Bangs Laboratories, Fishers,Ind.) was thoroughly resuspended and 2 ml aliquoted into a 15 ml conicaltube. The magnetic latex was suspended in 12 ml distilled water andseparated from the solution for 10 min using a magnet (PerSeptiveBiosystems, Framingham, Mass.). While maintaining the separation of themagnetic latex with the magnet, the liquid was carefully removed using a10 ml sterile pipette. This washing process was repeated an additionalthree times. After the final wash, the latex was resuspended in 2 ml ofdistilled water. In a separate 50 ml conical tube, 10 mg of avidin-HS(NeutrAvidin, Pierce, Rockford, Ill.) was dissolved in 18 ml of 40 mMTris, 0.15 M sodium chloride, pH 7.5 (TBS). While vortexing, the 2 ml ofwashed magnetic latex was added to the diluted avidin-HS and the mixturemixed an additional 30 seconds. This mixture was incubated at 45° C. for2 hr, shaking every 30 minutes. The avidin magnetic latex was separatedfrom the solution using a magnet and washed three times with 20 ml BBSas described above. After the final wash, the latex was resuspended in10 ml BBS and stored at 4° C.

Immediately prior to use, the avidin magnetic latex was equilibrated inpanning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH7.5). The avidin magnetic latex needed for a panning experiment (200μl/sample) was added to a sterile 15 ml centrifuge tube and brought to10 ml with panning buffer. The tube was placed on the magnet for 10 minto separate the latex. The solution was carefully removed with a 10 mlsterile pipette as described above. The magnetic latex was resuspendedin 10 ml of panning buffer to begin the second wash. The magnetic latexwas washed a total of 3 times with panning buffer. After the final wash,the latex was resuspended in panning buffer to the starting volume.

Example 4 Selection of Recombinant Polyclonal Antibodies to Bacillusanthracis SAP Antigen

Binding reagents that specifically bind to B. anthracis SAP wereselected from the phage display libraries created from hyperimmunizedmice as described in Example 3.

Panning

First round antibody phage were prepared as described in Example 3 usingBS45 uracil template. Electroporations of mutagenesis DNA were performedyielding phage samples derived from different immunized mice. To createmore diversity in the recombinant polyclonal library, each phage samplewas panned separately.

Before the first round of functional panning with biotinylated SAPantigen, antibody phage libraries were selected for phage displayingboth heavy and light chains on their surface by panning with7F11-magnetic latex (as described in Examples 21 and 22 of U.S. patentapplication Ser. No. 08/835,159, filed Apr. 4, 1997). Functional panningof these enriched libraries was performed in principle as described inExample 16 of U.S. patent application Ser. No. 08/835,159. Specifically,10 μL of 1×10⁻⁶ M biotinylated SAP antigen was added to the phagesamples (approximately 10⁻⁸ M SAP final concentration), and the mixtureallowed to come to equilibrium overnight at 2-8° C.

After reaching equilibrium, samples were panned with avidin magneticlatex to capture antibody phage bound to SAP. Equilibrated avidinmagnetic latex (Example 3), 200 μL latex per sample, was incubated withthe phage for 10 min at room temperature. After 10 min, approximately 9ml of panning buffer was added to each phage sample, and the magneticlatex separated from the solution using a magnet. After a ten minuteseparation, unbound phage was carefully removed using a 10 ml sterilepipette. The magnetic latex was then resuspended in 10 ml of panningbuffer to begin the second wash. The latex was washed a total of threetimes as described above. For each wash, the tubes were in contact withthe magnet for 10 min to separate unbound phage from the magnetic latex.After the third wash, the magnetic latex was resuspended in 1 ml ofpanning buffer and transferred to a 1.5 mL tube. The entire volume ofmagnetic latex for each sample was then collected and resuspended in 200ul 2xYT and plated on 150 mm LB plates as described in Example 3 toamplify bound phage. Plates were incubated at 37° C. for 4 hr, thenovernight at 20° C.

The 150 mm plates used to amplify bound phage were used to generate thenext round of antibody phage. After the overnight incubation, secondround antibody phage were eluted from the 150 mm plates by pipetting 10mL of 2xYT media onto the lawn and gently shaking the plate at roomtemperature for 20 min. The phage samples were then transferred to 15 mldisposable sterile centrifuge tubes with a plug seal cap, and the debrisfrom the LB plate pelleted by centrifuging the tubes for 15 min at 3500rpm. The supernatant containing the second round antibody phage was thentransferred to a new tube.

A second round of functional panning was set up by diluting 100 μL ofeach phage stock into 900 μL of panning buffer in 15 ml disposablesterile centrifuge tubes. Biotinylated SAP antigen was then added toeach sample as described for the first round of panning, and the phagesamples incubated for 1 hr at room temperature. The phage samples werethen panned with avidin magnetic latex as described above. The progressof panning was monitored at this point by plating aliquots of each latexsample on 100 mm LB agar plates to determine the percentage of kappapositives. The majority of latex from each panning (99%) was plated on150 mm LB agar plates to amplify the phage bound to the latex. The 100mm LB agar plates were incubated at 37° C. for 6-7 hr, after which theplates were transferred to room temperature and nitrocellulose filters(pore size 0.45 mm, BA85 Protran, Schleicher and Schuell, Keene, N.H.)were overlaid onto the plaques.

Plates with nitrocellulose filters were incubated overnight at roomtemperature and then developed with a goat anti-mouse kappa alkalinephosphatase conjugate to determine the percentage of kappa positives asdescribed below. Phage samples with lower percentages (<70%) of kappapositives in the population were subjected to a round of panning with7F11-magnetic latex before performing a third functional round ofpanning overnight at 2-8° C. using biotinylated SAP antigen atapproximately 2×10⁻⁹ M. This round of panning was also monitored forkappa positives. Individual phage samples that had kappa positivepercentages greater than 80% were pooled and subjected to a final roundof panning overnight at 2-8° C. at 5×10⁻⁹ M SAP. Antibody genescontained within the eluted phage from this fourth round of functionalpanning were subcloned into the expression vector, pBRncoH3.

The subcloning process was done generally as described in Example 18 ofU.S. patent application Ser. No. 08/835,159. After subcloning, theexpression vector was electroporated into DH10B cells and the mixturegrown overnight in 2xYT containing 1% glycerol and 10 μg/mltetracycline. After a second round of growth and selection intetracycline, aliquots of cells were frozen at −80° C. as the source forSAP polyclonal antibody production. Two polyclonal antibodies,designated IIT004.1 and IIT005.1, were selected from two librariesderived from different sets of spleens. Monoclonal antibodies wereselected from these polyclonal mixtures by plating a sample of themixture on LB agar plates containing 10 μg/ml tetracycline and screeningfor antibodies that recognized SAP.

Detection of Alkaline Phosphatase Conjugates

After overnight incubation of nitrocellulose filters on LB agar plates,filters were carefully removed from the plates with membrane forceps andincubated for 2 hr in 10 mM TRIS, 150 mM NaCl, 10 mM MgCl₂, 0.1 mMZnCl₂, 0.1% polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodiumazide, pH 8.0 (Block buffer). After 2 hr, the filters were incubatedwith goat anti-mouse kappa-AP (Southern Biotechnology Associates, Inc,Birmingham, Ala.) for 2-4 hours. The goat anti-mouse kappa-AP wasdiluted into Block buffer at a final concentration of 1 μg/ml. Filterswere washed three times with 40 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH7.5 (TBST) for 5 min each. After the final wash, the filters weredeveloped in a solution containing 0.2 M 2-amino-2-methyl-1-propanol(JBL Scientific, San Luis Obispo, Calif.), 0.5 M Tris, 0.33 mg/ml nitroblue tetrazolium ((NBT) Fisher Scientific, Pittsburgh, Pa.) and 0.166mg/ml 5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.

Expression and Purification of Recombinant Antibodies Against SAP

A shake flask inoculum was generated overnight from a −70° C. cell bankin an Innova 4330 incubator shaker (New Brunswick Scientific, Edison,N.J.) set at 37° C., 300 rpm. The inoculum was used to seed a 20 Lfermentor (Applikon, Foster City, Calif.) containing defined culturemedium (Pack et al. (1993) Bio/Technology 11: 1271-1277) supplementedwith 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,Detroit, Mich.), 12.5 g/L glycerol and 10 μg/ml tetracycline. Thetemperature, pH and dissolved oxygen in the fermentor were controlled at26° C., 6.0-6.8 and 25% saturation, respectively. Foam was controlled byaddition of polypropylene glycol (Dow, Midland, Mich.). Glycerol wasadded to the fermentor in a fed-batch mode. Fab expression was inducedby addition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L duringthe late logarithmic growth phase. Cell density was measured by opticaldensity at 600 μm in an UV-1201 spectrophotometer (Shimadzu, Columbia,Md.). Following run termination and adjustment of pH to 6.0, the culturewas passed twice through an M-210B-EH Microfluidizer (Microfluidics,Newton, Mass.) at 17,000 psi. The high pressure homogenization of thecells released the Fab into the culture supernatant.

The first step in purification was expanded bed immobilized metalaffinity chromatography (EB-IMAC). Streamline™ chelating resin(Pharmacia, Piscataway, N.J.) was charged with 0.1 M NiCl₂ and was thenexpanded and equilibrated in 50 mM acetate, 200 mM NaCl, 10 mMimidazole, 0.01% NaN₃, pH 6.0 buffer flowing in the upward direction. Astock solution was used to bring the culture homogenate to 10 mMimidazole, following which it was diluted two-fold or higher inequilibration buffer to reduce the wet solids content to less than 5% byweight. It was then loaded onto the Streamline column flowing in theupward direction at a superficial velocity of 300 cm/hr. The cell debrispassed through unhindered, but the Fab was captured by means of the highaffinity interaction between nickel and the hexahistidine tag on the Fabheavy chain. After washing, the expanded bed was converted to a packedbed and the Fab was eluted with 20 mM borate, 150 mM NaCl, 200 mMimidazole, 0.01% NaN₃, pH 8.0 buffer flowing in the downward direction.

The second step in the purification used ion-exchange chromatography(IEC). Q Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) wasequilibrated in 20 mM borate, 37.5 mM NaCl, 0.01% NaN₃, pH 8.0. The Fabelution pool from the EB-IMAC step was diluted four-fold in 20 mMborate, 0.01% NaN₃, pH 8.0 and loaded onto the IEC column. Afterwashing, the Fab was eluted with a 37.5-200 mM NaCl salt gradient. Theelution fractions were evaluated for purity using an Xcell II™ SDS-PAGEsystem (Novex, San Diego, Calif.) prior to pooling. Finally, the Fabpool was concentrated and diafiltered into 20 mM borate, 150 mM NaCl,0.01% NaN₃, pH 8.0 buffer for storage. This was achieved in a SartoconSlice™ system fitted with a 10,000 MWCO cassette (Sartorius, Bohemia,N.Y.). The final purification yields were typically 50%. Theconcentration of the purified Fab was measured by UV absorbance at 280nm, assuming an absorbance of 1.6 for a 1 mg/ml solution.

Culture of Bacillus spp. and Preparation of Cleared Culture SupernatantAntigen

Nonencapsulated Bacillus anthracis, Sterne strain was obtained from theColorado Serum Company. B. cereus OH599 was the kind gift of Dr. A.Kotiranta, B. globigii was obtained from Dr. L. Larson, and B.thuringiensis 10792 was obtained from the American Type CultureCollection (Manassas, Va.). Organisms were cultured on tryptic soy agarcontaining 5% sheep blood (Hardy Diagnostics, Santa Maria, Calif.) or inbrain heart infusion broth (Becton Dickinson and Company, Cockeysville,Md.) at 37° C.

For preparation of cleared culture supernatant antigens, B. anthracis,Sterne strain was grown in brain heart infusion broth at 37° C. withaeration for 24 h. A sample of the culture was serially diluted 10-foldin sterile 0.01 M phosphate buffered saline, pH 7.4 (PBS) and 100 μl ofeach dilution was plated on a blood agar plate for determination of thenumber of viable organisms. The culture was subjected to centrifugationat 10,000×g for 20 min at 4° C. using a J2-21 centrifuge (Beckman,Fullerton, Calif.). The supernatant was transferred to a sterile bottleand a protease inhibitor cocktail (Sigma-Aldrich, Inc) was added. Thesample was filtered using a 0.2 μm pore-size membrane filter unit(Millipore Corp., Bedford, Mass.) then dialyzed in 4 L of 0.01M PBS, pH7.4, 2 mM EDTA, 0.1 mM phenymethylsulfonyl fluoride at 4° C. with fourbuffer changes in 24 h. The dialyzed sample was aliquoted and stored at−80° C. The concentration of SAP in the cleared culture supernatant wasquantified by a sandwich enzyme-linked immunosorbant assay usingpurified recombinant SAP as a standard. Analysis of the amount of SAPrecovered from the culture supernatant indicated that 1 ng of SAPcorresponded to approximately 2.9×10³ organisms (i.e. 0.35 pg/organism).

Example 5 Selection of Monoclonal Antibodies to Sap from the RecombinantPolyclonal Antibody Mixtures

Monoclonal antibodies against SAP were isolated from clones containingthe recombinant polyclonal mixtures (Example 4) by plating a dilutedsample of the mixture on LB agar plates containing 10 μg/mltetracycline. Individual colonies were then tested for the ability toproduce antibody that recognized recombinant SAP using surface plasmonresonance (BIACORE) (BIACORE, Uppsala, Sweden). Small scale productionof these monoclonal antibodies was accomplished using a Ni-chelatebatch-binding method (see below). Antibodies isolated from this methodwere diluted 1:3 in HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.005% polysorbate 20 (v/v)), captured with a goat anti-mousekappa antibody (Southern Biotechnology Associates, Inc, Birmingham,Ala.) coupled to a BIACORE CM5 sensor chip, and tested for the abilityto bind recombinant SAP. Antibodies that bound SAP were then evaluatedusing BIACORE epitope mapping analysis. Antibodies that bound distinctepitopes were produced on a larger scale and then conjugated to biotinand alkaline phosphatase. These conjugates were then tested in an ELISAassay to determine the sensitivity and utility of these antibodies.

Minipreparation of Monoclonal Antibodies by Ni-Chelate Batch-BindingMethod

Individual colonies were isolated from the recombinant polyclonalmixtures (Example 4) and used to inoculate 3 ml cultures of 2xYT mediumcontaining 1% glycerol supplemented with 10 μg/ml tetracycline. Thesecultures were grown in an Innova 4330 incubator shaker (New BrunswickScientific, Edison, N.J.) set at 37° C., 300 rpm. The next morning 0.5ml of each culture was used to inoculate shake flasks containing 50 mlof defined medium, (Pack et al. (1993) Bio/Technology 11: 1271-1277)supplemented with 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L caseindigest (Difco, Detroit, Mich.), 12.5 g/L glycerol and 10 μg/mltetracycline. These cultures were shaken at 300 rpm, 37° C. until anoptical density of 4 was reached at 600 nm. Fab expression was theninduced by adding L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L andshifting the temperature to 23° C. with overnight shaking. The next daythe following was added to the 50 ml cultures: 0.55 ml of 1 M imidazole,5 ml B-PER (Pierce, Rockford, Ill.) and 2 ml Ni-chelating resin(Chelating Sepharose FastFlow® resin Pharmacia, Piscataway, N.J.). Themixture was shaken at 300 rpm, 23° C. for 1 hour after which timeshaking was stopped and the resin allowed to settle to the bottom of theflasks for 15 minutes.

The supernatant was then poured off and the resin resuspended in 40 mlof BBS (20 mM borate, 150 mM NaCl, 0.1% NaN₃, pH 8.0) containing 10 mMimidazole. This suspension was transferred to a 50 ml conical tube andthe resin washed a total of 3 times with BBS containing 10 mM imidazole.Washing was accomplished by low speed centrifugation (1100 rpm for 1minute), removal of supernatant and, resuspension of the resin in BBScontaining 10 mM imidazole. After the supernatant of the final wash waspoured off, 0.5 ml of 1 M imidazole was added to each tube, vortexbriefly, and transferred to a sterile microcentrifuge tube. The sampleswere then centrifuged at 14 krpm for 1 minutes and the supernatanttransferred to a new microcentrifuge tube. Antibodies contained in thesupernatant were then analyzed for binding to SAP using a BIACORE(BIACORE, Uppsala, Sweden).

Selection and Cloning of a Recombinant Polyclonal Antibody Complementaryto IIT005.1.13 and IIT005.1.C.11 Monoclonal Antibodies

A monoclonal antibody designated IIT004.1.12 was selected from thepolyclonal library designated IIT004.1, biotinylated, and 15 μl of a10⁻⁶ M solution was mixed with soluble recombinant SAP antigen (7.5 μlof a 10⁻⁷M solution). This mixture was incubated for 15 minutes at roomtemperature. Fifteen microliters of each mixture was added to 50 μl ofphage library IIT005.1 diluted in 1 ml panning buffer (40 mM Tris, 150mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH 7.5) and incubated overnight at2-8° C. Final concentrations were 10⁻⁸ M biotinylated monoclonalantibody and 5×10⁻¹⁰ M SAP. The sample was panned with avidin magneticlatex and plated as described in Example 4. The eluted phage weresubjected to another round of selection using these conditions and theresulting polyclonal library was designated IIT005.1.C. Two monoclonalantibodies designated IIT005.1.13 and IIT005.1.C.11 were selected fromthe IIT005.1 and IIT005.1.C libraries respectively, biotinylated andcomplexed with SAP using the conditions described above. Complementarypolyclonal antibodies were selected as described above from phagelibrary IIT005.1 using monoclonal antibodies IIT005.1.13 andIIT005.1.C.11. These complementary polyclonal antibodies were designatedIIT005.1.13.1 and IIT005.1.C.11.1 and were subcloned as described inExample 18 of U.S. patent application Ser. No. 08/835,159.

Example 6 Specificity of Monoclonal/Polyclonal Antibodies to SAPDetermined by Western Blot and Indirect Immunofluorescence Analysis

The specificity of monoclonal and polyclonal antibodies against B.anthracis, Sterne strain SAP was visualized by Western blot analysis.Recombinant antibodies against SAP were tested for reactivity torecombinant SAP as well as to SAP isolated from the cleared culturesupernatant of Bacillus anthracis Sterne strain. Cross reactivity toother Bacillus strains was also tested. Culture supernatant proteins andwhole cell lysates of B. anthracis, Sterne strain, B. cereus OH599, B.globigii and B. thuringiensis 10792 equivalent to 10⁸ organisms wereseparated by electrophoresis in 4-20% TRIS-glycine SDS-polyacrylamidegels (Novex, San Diego, Calif.) under reducing conditions. The proteinswere transferred to ProBlott™ membranes (Applied Biosystems, FosterCity, Calif.) using 10 mM CAPS/10% methanol transfer buffer. Themembranes were blocked in 10 mM TRIS, 150 mM NaCl, 10 mM MgCl₂, 0.1 mMZnCl₂, 0.1% polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodiumazide, pH 8.0 (Block buffer) for 1 h at room temperature.

The membranes were then incubated in 5 μg/ml of monoclonal orrecombinant polyclonal antibody diluted in Block buffer for 1 h and thenwashed three times with 40 mM TRIS, 150 mM NaCl, 0.05% Tween 20, pH 7.5(TB ST) (Fisher Chemical, Pittsburgh, Pa.) for 5 min each. Afterwashing, the membranes were incubated in rabbit anti-mouse IgG(H&L)-alkaline phosphatase conjugate (Southern Biotechnology, Inc,Birmingham, Ala.) diluted 1:1000 in Block buffer. The membranes werewashed three times with TBST for 5 min each and developed in a solutioncontaining 0.2 M 2-amino-2-methyl-1-propanol (JBL Scientific, San LuisObispo, Calif.), 0.5 M TRIS, 0.33 mg/ml nitro blue tetrazolium ((NBT)Fisher Scientific, Pittsburgh, Pa.) and 0.166 mg/ml5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.

The anti-SAP recombinant polyclonal antibodies reacted with recombinantSAP, SAP protein isolated from the culture supernatant, and the cellpellet of B. anthracis, Sterne strain. The antibodies did not react withany proteins in the culture supernatant or cell pellet of the otherBacillus species tested (B. cereus and thuringiensis). A goatanti-anthrax polyclonal serum was used to demonstrate cross-reactivityof B. anthracis antibodies with proteins of other Bacillus species (datanot shown). Conjugates alone served as negative controls.

The specificity of antibodies against B. anthracis, Sterne strain wasalso tested by indirect immunofluorescence. Localization of SAP to theouter membrane of unencapsulated B. anthracis, Sterne strain wasdemonstrated using an indirect immunofluorescence technique. B.anthracis, B. cereus, and B. thuringiensis were washed and resuspendedin PBS to yield 1×10⁸ organisms per ml. Four microliters of thesuspensions were applied to wells of an eight well microscope slide andallowed to air dry. The slides were lightly heated to fix the smears tothe slide and covered with 0.1 mg/ml of antibody diluted in PBScontaining 1% BSA. The smears were incubated with antibody for 1 h at37° C. in a moist chamber. After washing the slides three times bysoaking in PBS for 5 min each, the smears were covered with fluoresceinisothiocyanate-conjugated rabbit anti-mouse IgG (H&L) F(ab′)₂ (ZymedLaboratories, Inc., South San Francisco, Calif.) diluted 1:80 in PBS, 1%BSA, 0.05% Evans Blue (Sigma). The slides were incubated for 1 h at 37Cin a moist chamber then washed as described above. After a final wash indeionized water, the slides were allowed to air dry in the dark.Coverslips were mounted using a 90% glycerol mounting medium containing10 mg/mlp-phenylenediamine, pH 8.0.

The slides were examined for fluorescent organisms using anepifluorescence microscope with a 63× objective lens (Leitz WetzlerGermany). The recombinant polyclonal antibody (ITT005.1) demonstrated 4+fluorescence with unencapsulated B. anthracis and did not react with B.cereus, or B. thuringiensis. Negative controls includedfluorescein-conjugated antibody alone, and a murine polyclonal antiserumspecific for B. anthracis, Sterne strain spore coat proteins.

Example 7 Sensitivity and Specificity of an ELISA Plate Assay forDetection of B. anthracis SAP

This Example demonstrates that an ELISA assay using the reagents andmethods of the invention are not only highly sensitive for B. anthracis,but are also highly specific for this particular Bacillus species.

The sensitivity and specificity of various monoclonal/recombinantpolyclonal antibody pairs were determined by performing a sandwich assayusing biotinylated monoclonal antibodies and alkalinephosphatase-conjugated recombinant polyclonal antibodies. Assays wereperformed with NeutraAvidin or streptavidin coated plates, such asReacti-Bind™ streptavidin coated polystyrene 96 well plates (PierceChemical, Rockford, Ill.). After washing the 96 well plate with BBS (20mM borate, 150 mM NaCl, 0.01% NaN₃, pH 8.0) containing 0.02% TWEEN-20,biotinylated monoclonal antibodies (50 μL of 2.5 μg/mL diluted in Blockbuffer (10 mM Tris, 150 mM NaCl, 10 mM MgCl₂, 0.1 mM ZnCl₂, 0.1%polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodium azide, pH 8.0))were added to the wells. The plate was incubated at room temperature for1 hr.

The plate was then washed, after which various dilutions (10 ng/ml to0.625 ng/ml) of soluble SAP antigen (50 μL of recombinant SAP or SAP inculture supernatants (as prepared in Example 4) were added in duplicateto the biotinylated monoclonal wells. The plates were incubated for onehour at room temperature or overnight at 2-8° C., after which the platewas washed. The appropriate recombinant polyclonal antibody-alkalinephosphatase conjugate (50 μL of 2.5 μg/mL diluted in Block) was addedand incubated at room temperature for 1 hr. After 1 hr, the plate waswashed and developed using the ELISA Amplification System (Gibco BRL,Gaithersburg, Md.) according to the manufacturer's instructions.

Results from several assays are compiled in accompanying tables 2-5.These data indicate that the assays can detect less than 0.625 ng of SAPprotein. This amount of SAP corresponds to approximately 1.8×10³Bacillus anthracis organisms per ml. Significantly, little or no crossreactivity to other related Bacillus species was detected.

TABLE 2 IIT005.1.C.11-BIOTIN WITH IIT005.1.C11.1-AP B. anthracisUndiluted culture SAP Bacillus (cfu/ml) A490 (ng/mL) A490 species A49028330 3.4 10 3.55 cereus 0.28 14165 2.8 5 3.45 thuringiensis 0.27 70832.14 2.5 2.94 subtilis niger 0.55 3541 1.56 1.25 2.01 subtilis 0.51 17701.17 0.625 1.51 BHI broth 0.48 0 0.92 0 0.92 media

TABLE 3 IIT005.1.13-BIOTIN WITH IIT005.1.13.1-AP B. anthracis Undilutedculture Bacillus (cfu/ml) A490 species A490 28330 3.13 cereus 0.28 141652.21 thuringiensis 0.27 7083 1.5 subtilis niger 0.48 3541 0.99 subtilis0.5 1770 0.78 BHI broth 0.423 0 0.55 media

TABLE 4 IIT005.1.C.11-BIOTIN WITH IIT005.1-AP B. anthracis Undilutedculture SAP Bacillus (cfu/ml) A490 (ng/mL) A490 species A490 28330 2.8710 3.4 cereus 0.09 14165 1.698 5 2.56 thuringiensis 0.14 7083 1 2.5 1.49subtilis niger 0.13 3541 0.55 1.25 0.82 subtilis 0.14 1770 0.35 0.6250.49 BHI broth 0.148 0 0.14 0 0.19 media

TABLE 5 IIT005.1.13-BIOTIN WITH IIT005.1-AP B. anthracis Undilutedculture Bacillus (cfu/ml) A490 species A490 28330 1.77 cereus 0.08514165 0.99 thuringiensis 0.121 7083 0.54 subtilis niger 0.125 3541 0.34subtilis 0.124 1770 0.23 BHI broth 0.125 0 0.14 media

These results demonstrate that four different monoclonal/recombinantpolyclonal antibody preparations exhibit great sensitivity for B.anthracis while not cross reacting with other Bacillus species.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. An isolated antibody that specifically binds to the surface arrayprotein set forth in SEQ ID NO:1 but lacks cross-reactivity withBacillus species other than B. anthracis.
 2. The antibody of claim 1,wherein the antibody is a recombinantly-produced antibody.
 3. Theantibody of claim 1, wherein the antibody is a recombinantly-producedpolyclonal antibody.
 4. The antibody of claim 1, wherein the antibody isa monoclonal antibody.
 5. The antibody of claim 1, wherein the antibodyis an Fab fragment.
 6. The antibody of claim 1, wherein the antibody isan Fab′ fragment.
 7. The antibody of claim 1, wherein the antibody is asingle chain antibody.
 8. The antibody of claim 1, wherein the antibodyis displayed as a fusion protein on a surface of a replicable geneticunit.
 9. The antibody of claim 8, wherein the replicable genetic unit isa filamentous phage.
 10. The antibody of claim 1, wherein the antibodyis biotinylated.
 11. The antibody of claim 1, wherein the antibody isattached to a detectable label.
 12. The antibody of claim 1, wherein theantibody is immobilized on a solid support.
 13. The antibody of claim11, wherein the solid support is a porous member.